When examining a sample, it is often important to be able to rapidly form an image of entire sample volumes. To do so, the use of an SPIM microscope is considered in particular.
The SPIM (single plane illumination microscopy) technique, in which a sample is illuminated in layers, allows image data to be captured more quickly and in a gentler manner for the sample compared with scanning a sample at specific points, for example. A known field of use of SPIM technology is the field of fluorescence microscopy, in which fluorophores in the sample are excited by means of laser light. In SPIM technology, excitation takes place only in one of an illumination light sheet. Damage to the sample caused by illumination light in other planes is thus avoided.
An optical apparatus that functions in accordance with the SPIM method is described in DE 102 57 423 A1. In this microscope, a sample is illuminated by a thin light sheet whilst being observed perpendicularly to the plane of the illuminating light sheet. Here, the illumination and the detection take place over two separate optical ray paths each having separate optics, in particular having two separate objectives perpendicular to one another.
DE 10 2009 044 983 A1 discloses a microscope which includes an illumination device by which a light sheet for illuminating a sample region is generated, which light sheet is extended in an approximately planar manner in the direction of an illumination axis of an illumination ray path and in the direction of a transverse axis arranged transversely to the illumination axis. The microscope additionally includes a detection device by which light radiated from the sample region along a detection axis of a detection ray path is detected, the illumination axis and detection axis and also the transverse axis and detection axis being arranged at an angle other than zero to one another, and the detection device additionally including a detection objective in the detection ray path. In a microscope of this kind, the detection device additionally includes an optical detection element, which is arranged spatially separately from a front lens of the detection objective, is adjustable independently thereof, and by means of which the size of a detection image field can be continuously varied, and/or by means of which a detection focus plane in the sample region can be continuously adjusted.
From Fahrbach, F. O., Voigt, F. F., Schmid, B., Heimchen, F. & Huisken, J. Rapid 3D light-sheet microscopy with a tunable lens. Opt. Express 21, 21010 (2013), it is known to rapidly shift a light sheet along the detection axis in order to rapidly form an image of volumes, and to correct the sharpness plane of the detection optics using a tunable lens.
DE 10 2013 205 115 A1 discloses an SPIM arrangement including an illumination device for generating a light sheet that illuminates a sample, and including a detection device that has a detector and detects detection light emanating from the sample. In terms of efficient and gentle sample examination, the SPIM arrangement is equipped with structurally simple means and is developed such that the detection device includes a device for assigning various focal planes of the light sheet to various regions of the detector. The device can, for example, include a multi-focus grating or a chromatic correction grating.